The pressing needs in translating recent achievements of genomic and proteomic screens ex vivo into the visualization of markers in living systems necessitate the development of novel molecular biomarker imaging techniques. The ability to apply molecular imaging to novel markers in vivo would have significant implications for early detection of disease, assessing patient-specific therapies and monitoring dynamic changes in expression patterns during disease progression. This application builds on our recent innovations in designing, testing and applying enzyme-mediated MR signal amplification strategy (MRamp) for imaging molecular targets. The proposed research is based on our observation that paramagnetic phenols in the presence of oxidoreductases give markedly enhanced relaxivity and MR signal. We previously proposed to harness MRamp effect and apply it for the needs of MR molecular imaging. As a result, we accomplished a transition of our research from in vitro level to in vivo experiments. Reaching the following milestones were instrumental in achieving the aims of the research: 1) designing and scaling up synthesis of MRamp substrates;2) determining that the nature of aromatic reducing group linked to paramagnetic moiety defines substrate specificity and enzyme selectivity;3) providing evidence that MRamp mechanism includes both polymerization and binding of paramagnetic oligomers to macromolecules;4) optimizing a complete protocol for small conjugates of anti-receptor antibody and amplification enzymes ( binary amplification system );5) performing the testing of the developed system by using MRI in EGFR-expressing tumors;6) testing alternative amplification enzymes and identifying a candidate for future research. By building on the above key findings we propose to achieve the following specific aims: Specific Aim. 1. Optimize and test in vivo single-enzyme targeted amplification imaging system. We hypothesize that MR signal amplification strategy could be improved by optimizing in vivo delivery. This can be achieved by using a) single, phenol- oxidizing enzyme covalently linked to F(ab')2 fragment of EGFR antibody. This hypothesis will be tested in EGFR-overexpressing tumor models. Specific Aim 2. Develop and test two-enzyme, bi-specific approach for imaging tumor cells co-expressing two different molecular markers. We hypothesize that by using two antibody fragments directed against different targets on the same cells we will image co-expression of adhesion molecule (EpCAM) and EGF receptor on non-small cell lung cancer (NSCLC) cells that are likely to respond to combined antibody therapy. This hypothesis will be tested in a model of NSCLC metastasis to the brain. Specific Aim 3. To use MRamp strategy for imaging inflammatory response and receptor repertoire of tumors in vivo. We hypothesize that monocyte/neutrophil component of inflammatory response can be imaged in tumors separately from receptors by using two MRamp substrates: the first having narrow myeloperoxidase specificity, and the second having laccase specificity. This hypothesis will be tested in transgenic model of prostate cancer. Public health relevance statement: The development of new drugs that can efficiently eliminate tumor cells or slow heart disease and diabetes requires ample testing in laboratory animals to prove safety and efficacy. The use of medical scanners that detect these cellular processes with high accuracy in live animals has the potential to significantly decrease the time between discovery of and subsequent clinical use of new medicines. This decreased time benefits both patients and taxpayers. We are proposing research approaches that will lead to the development of new tools (imaging drugs and compositions) for use with medical scanners. These tools will have applications for tracking the molecules that are linked to the abnormal cells. This research will help scientists and physicians to detect these cells and follow their response to medicines.